EP0534878A1 - Molécules d'ADN recombinants contenant la partie codante d'une chaîne Vbêta17 du récepteur pour l'antigène des cellules T et leurs utilisations - Google Patents

Molécules d'ADN recombinants contenant la partie codante d'une chaîne Vbêta17 du récepteur pour l'antigène des cellules T et leurs utilisations Download PDF

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EP0534878A1
EP0534878A1 EP92430022A EP92430022A EP0534878A1 EP 0534878 A1 EP0534878 A1 EP 0534878A1 EP 92430022 A EP92430022 A EP 92430022A EP 92430022 A EP92430022 A EP 92430022A EP 0534878 A1 EP0534878 A1 EP 0534878A1
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Prior art keywords
sequence
chain
recombinant dna
human
coding
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German (de)
English (en)
French (fr)
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François Romagne
André van Agthoven
Bernard Malissen
Lydia Besnardeau
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Immunotech Sa SA
Immunotech SAS
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Immunotech Sa SA
Immunotech SAS
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence

Definitions

  • the present invention relates to new recombinant DNA molecules coding in particular for a V ⁇ 17 chain of the receptor for the T cell antigen, their process of preparation, antibodies and the drugs containing them.
  • TCR human T-cell antigen
  • T cells The easy study of the imbalance between the sub-populations would then allow the differential diagnosis of diseases causing proliferation of T cells.
  • the latter could also be the witness of a specific immune response against a particular tissue, pathogenic such as a tumor by example. It is known in particular that antibodies against the CD4 and CD8 antigens which distinguish two populations of T cells, are witnesses of a series of immune diseases including AIDS.
  • the Applicant has discovered new recombinant DNAs enabling the preparation of new eukaryotic expression vectors which were remarkable transfection agents for eukaryotic cells, which transfected cells were excellent immunogens leading to antibodies, in particular monoclonal antibodies, directed towards the V ⁇ 17 chain human TCRs.
  • recombinant DNA is meant an artificially created nucleotide sequence.
  • chimera By chimera is meant that the recombinant DNA comprises at least two sequences originating from different origins, for example 3, 4 or 5 and preferably 2 sequences from two different origins.
  • sequences come from two different species and the origin of one of them is murine; the other (or the others) will come in particular from a primate and preferably from humans.
  • the ⁇ receptor for T cells is a heterodimeric protein composed of two polypeptide chains, ⁇ and ⁇ , linked by a disulfide bridge.
  • the receptor is expressed on the surface of T cells in association with the CD3 complex.
  • the locus corresponding to a particular TCR chain includes a large selection of distinct gene segments, including many different V segments, several J segments and one or two constant C segments. These segments may undergo somatic rearrangements during thymic maturation of T cells thus producing a single gene, which is transcribed and containing a V segment, a J segment, a C segment and, where appropriate, a D segment between the V and J segments.
  • the entire sequence coding for the C ⁇ part can come from a murine and in this case the sequence of the other human does not include any nucleotide corresponding to the C ⁇ part.
  • the sequence originating from a human comprises a short C ⁇ part, preferably less than 100 nucleotides and in particular approximately 60 nucleotides; therefore, only a large part C ⁇ of the chimera comes from the murine (this part being the complementary part absent from the human sequence above) and the rest coming from man.
  • the two sequences of different origins are linked at the level of a selected restriction site, in phase in the reading frame.
  • the human sequence will necessarily include, in addition to the V ⁇ 17, J ⁇ and D ⁇ parts and a part of C ⁇ , the entire leader sequence.
  • the selected restriction site can be naturally present on each of the two sequences or naturally present on only one of the two sequences, said restriction site being artificially created on the other sequence.
  • the above restriction site must be unique on the segment C ⁇ 1 or murine C ⁇ 2 and preferably unique in the human segment.
  • the selected restriction site When the selected restriction site is created artificially, it can be produced for example by deletion or addition of nucleotides or preferably by mutation according to the methods known per se, in particular by site-directed mutagenesis.
  • the DNA damage thus produced allows the introduction of a normally absent restriction site.
  • a BglII site naturally present at the level of the DNA coding for the human C ⁇ 1 and C ⁇ 2 region, but absent from the gene coding for the mouse TCR chain, can be introduced into the mouse C ⁇ 1 or C ⁇ 2 gene. by site-directed mutagenesis, but possibly mutagenesis by chain polymerization (PCR).
  • PCR chain polymerization
  • the restriction site thus created will preferably be placed at a site homologous to the site at which the restriction site is naturally present in humans, so as to allow cleavage, both of the sequence murine as human sequence using said enzyme, and then, by phase ligation in the reading frame, create the desired chimera. From the above, it is understood that if one chooses a given restriction enzyme, a single corresponding restriction site must at most be present on the C ⁇ 1 or murine C ⁇ 2 segment and preferably unique on the human segment and moreover, at the level of the sequence coding for the C ⁇ 1 or C ⁇ 2 part, at a location allowing the transcription in phase of a DNA coding in particular the TCR chain.
  • Preferred recombinant DNAs according to the invention are characterized in that the restriction site is natural on the sequence coding for human part C.
  • Chimeras very particularly preferred according to the invention have a human part characterized in that it is the sequence coding for V ⁇ 17, D ⁇ , J ⁇ human segments, and a short C ⁇ part up to the unique BglII site of C ⁇ .
  • the murine part of the notable chimeras according to the invention is characterized in that the restriction site, in particular BglII, is artificially created on the chain of murine origin.
  • the recombinant DNA according to the invention may be in the form of a single or single-stranded nucleotide sequence, limited to parts V, D, J and C, above, or comprising other nucleotides, for example and in particular those constituting the head sequence.
  • the above sequence can in particular be included in an expression vector, and in this case preferably in a eukaryotic expression vector.
  • the above sequence can also be included with a view to its replication in another vector, for example in a plasmid such as for example BLUESCRIPT.
  • a eukaryotic expression vector mention may very particularly be made of pH ⁇ Pr1-Neo. Mention may also be made of pH ⁇ Pr1gpt and generally any eukaryotic expression vector possessing the promoter regulatory signals and a polyadenylation signal allowing cDNA expression.
  • the choice of the vector-host couple is within the competence of a person skilled in the art.
  • Recombinant DNAs which are very particularly preferred according to the invention are characterized in that they are in the form of a eukaryotic expression vector, which is preferably derived from pH ⁇ Pr1-Neo.
  • the above recombinant DNA in particular in the form of a eukaryotic expression vector, can be used to transfect into a suitable host the sequence coding for TCR.
  • Host cells thus transfected optionally treated, for example previously transfected with the CD3 Zeta chain to obtain better expression, can be used as immunogen, preferably in mice.
  • the present application also relates to antibodies, in particular monoclonal antibodies, characterized in that they are prepared by use of a recombinant DNA described above.
  • the present invention also relates to anti-V ⁇ 17 chain antibodies of the human T cell receptor.
  • the above antibodies have remarkable properties because they are perfectly characterized with respect to the ⁇ chains of TCR, and in particular with respect to the variable regions V ⁇ 17 of the chains of TCR, but also with respect to the D ⁇ and J ⁇ regions in their typical rearrangement.
  • These antibodies are therefore capable of distinguishing V ⁇ 17 elements from the population of human T cells and thus specifically recognizing, for example a given disease or deficiency.
  • anti-variable part antibodies are preferred, in particular V ⁇ 17, which have remarkable properties both for diagnosis and for therapy, curative or preventive, in humans or animals, of autoimmune diseases and septic diseases, in particular those due to mycobacteria and in particular to staphylococci.
  • the antibodies according to the invention can for this purpose be used directly or, if desired, coupled according to the methods known per se, for example using a heterobifunctional agent such as SMCC, to a toxin or an active principle cytotoxic to which T cells are sensitive
  • compositions characterized in that they contain at least one of the antibodies as defined above.
  • compositions can be presented in particular in parenteral, in particular injectable forms, commonly used in human medicine, for example for the subcutaneous, intramuscular or intravenous route.
  • the above antibodies can also be used to treat immune diseases or those causing proliferation of T cells carrying the V ⁇ 17 chains, as well as leukemia with T cells carrying the V ⁇ 17 chain, by injection of the above antibodies.
  • the above antibodies can also be coupled to radioactive products, according to methods known in themselves (Chelating agents for metals such as Indium, Iodine substitution, etc.).
  • the antibodies according to the invention find their use in particular in the treatment of autoimmune diseases such as rheumatoid arthritis, diabetes, Sjögren's disease or lupus erythematosus.
  • the usual dose which varies according to the product used, the subject treated and the condition in question, can for example be 0.001 to 25 mg per day per kg of body weight in humans of a monoclonal antibody according to the invention coupled to ricin A as a cytotoxic, and preferably from 0.05 to 0.2 mg / kg / day.
  • the present invention also relates to use of said antibodies for the detection, identification or assay of the above antigens, as well as diagnostic compositions (or diagnostic products, intended for such visualization).
  • the above antibodies also find their use in the purification of products containing the antigenic part corresponding to TCR chains.
  • the above antibodies can be used for diagnostic purposes in cytofluorimetry and for this purpose can be coupled, by methods known per se, to fluorescent markers such as fluorescein isothiocyanate (FITC) or phycoerythrin, a more technical advantageous than the PCR study.
  • fluorescent markers such as fluorescein isothiocyanate (FITC) or phycoerythrin
  • the antibodies described above can also be labeled using enzymes. Mention may be made, for example, of glucose 6 phosphate dehydrogenase, acetylcholinesterase, glucose oxidase or betagalactosidase.
  • the above total antibodies can also be coupled to a radiolabel such as indium 111, for use in the imaging of T cell populations comprising the V ⁇ 17 region, in the diseased parts of the human body.
  • a radiolabel such as indium 111
  • the method described above is characterized in that, in addition, a eukaryotic cell is transfected comprising all the elements allowing the synthesis of the T cell antigen receptor, except for the chain homologous to the chain coded by the above chimaeric sequence, uses as cells the cells thus transfected in the corresponding murine strain, and fuses spleen cells taken from the murines to which the antigen has been administered to myeloma cells for prepare, according to the methods known per se, antibodies directed against the product of the human variable sequence.
  • the process described above can in particular be carried out as follows:
  • the nucleotide sequence coding for the V ⁇ 17, D ⁇ and J ⁇ parts of a human TCR chain and in particular a linear double strand DNA comprising the sequence coding for the V ⁇ 17, J ⁇ , D ⁇ and possibly C ⁇ parts in part of a TCR chain human, as well as its head gene, is obtained by cleavage using two different restriction enzymes : the enzyme corresponding to the restriction site selected at the C ⁇ part, for example BglII and another enzyme cleaving the sequence on the opposite side, after the leader gene, for example EcoRI from a double DNA sequence linear strand comprising said sequence.
  • the DNA thus obtained is ligated to a vector treated with the same two restriction enzymes, this vector comprising the sequence coding for the C ⁇ 2 (or C ⁇ 1) segment of the murine TCR chain, and the same restriction sites as above. , for example BglII and EcoRI.
  • the expected chimeric gene is thus obtained. However, it cannot be expressed as is. This is why on the one hand the vector comprising the chimera is treated with two restriction enzymes cutting on either side of the chimeric gene coding for the chimeric TCR; on the other hand, a eukaryotic expression vector is treated with the same two restriction enzymes.
  • a new chimeric eukaryotic expression vector is obtained.
  • Said eukaryotic expression vector must contain the elements necessary to ensure the correct transcription of the chimeric gene coding for the TCR.
  • a vector pH ⁇ Pr1-Neo which can be split by BamHI and SalI, and in particular comprises the gene for resistance to neomycin used as a selection marker, a eukaryotic promoter sequence for the ⁇ gene. actin, as well as a polyadenylation signal near the BamHI site.
  • the new chimeric vector obtained is advantageously used to transfect eukaryotic cells, preferably mouse T cells, in particular cell lines comprising all the elements for the expression of TCR with the exception of the gene provided by the chimeric vector.
  • eukaryotic cells preferably mouse T cells
  • cell lines comprising all the elements for the expression of TCR with the exception of the gene provided by the chimeric vector.
  • Such cells allow an easy selection of the cells expressing the chimera which alone are capable of expressing the TCR on the surface of the cell.
  • each ligation (above and below) is followed by transfection of an appropriate host and selection of cells containing the desired chimera.
  • the transfected cells can be administered as an antigen preferably to mice, for example by intraperitoneal injection. It is thus possible to take spleen cells from the murins thus treated, cells capable of secreting the desired antibodies in order to merge them with immortal cells, such as myeloma.
  • the selected hybridomas producing the desired antibodies thus allow the preparation of antibodies, in particular monoclonal antibodies directed against the V ⁇ 17 chain of the human TCR.
  • the double-stranded DNA coding for the human T cell antigen receptor chain can for example be prepared as follows: The study of the sequence of the genes coding for the V ⁇ 17 chain makes it possible to select a restriction site already present on a constant portion C ⁇ , or then leads to creating on said sequence such a site and allows the synthesis of new oligonucleotides which can be used to synthesize the Complementary DNA from messenger RNA.
  • the mRNA encoding a given TCR chain is purified from human T lymphocytes.
  • the complementary DNA is synthesized using an oligonucleotide, for example OHC ⁇ , specific for a given human gene and possibly containing the unique restriction site selected using a reverse transcriptase, synthesizes the second strand from a chain specific oligonucleotide, for example OL17, using a polymerase, and amplifies the gene obtained by polymerization chain reactions (PCR) by performing for example thirty cycles.
  • PCR polymerization chain reactions
  • a large amount of linear double-stranded DNA encoding is thus obtained. It is then possible to cut the appropriate enzymes, for example BglII and EcoRI, the fragments produced by PCR, so as to eliminate all or part of the sequence coding for the C ⁇ part, from the selected site and so as to conserve the V ⁇ 17 sequences. , D ⁇ and J ⁇ , as well as the entire head sequence.
  • appropriate enzymes for example BglII and EcoRI
  • the nucleotide sequence of murine origin comprising the complementary part of the C ⁇ part absent from the human sequence above, in the case where it is in the form of a vector comprising said sequence, can be prepared as follows : A complete cDNA is isolated containing all the coding part of the ⁇ chain of the TCR chosen from a cDNA library originating from a murine cell line, for example KB5C20, using a known probe; this cDNA is cloned into an appropriate plasmid, for example PUC19, then the resulting plasmid, called in this case pUCmC ⁇ 2, is transfected into a host, for example the bacterial strain DH1 of E. coli.
  • pUCmC ⁇ 2 can be digested with suitable restriction enzymes, for example EcoRI and HindIII, so as to obtain three fragments in the above case.
  • suitable restriction enzymes for example EcoRI and HindIII.
  • the fragment containing the entire coding sequence of the desired murine C ⁇ 2 (or C ⁇ 1) chain is purified (in the case of the abovementioned enzymes, EcoRI cuts from part V of the sequence).
  • EcoRI cuts from part V of the sequence.
  • the said sequence is then ligated with an appropriate phage, for example M13mp18, opened with the same restriction enzymes.
  • phage DNA exists in two forms: single strand for the encapsidated phage and double strand for its replicative form.
  • An oligonucleotide complementary to the region to be mutated, except for the desired mutation, is hybridized with the single-stranded form of the phage.
  • the complementary strand is synthesized in vitro by a DNA polymerase using the same oligonucleotide to initiate the reaction.
  • the new strand is closed on itself using a ligase.
  • the completely homologous double strand is then transfected, for example, into E. coli, in particular JM101.
  • the mutant colonies are selected, for example by hybridization with a radiolabeled oligonucleotide.
  • the double-stranded DNA of a subclone comprising the desired mutation is purified and the remainder of the sequence coding for the C ⁇ 2 (or C ⁇ 1) part has remained intact, and is cut by appropriate enzymes, for example EcoRI and EcoRV to isolate the fragment containing the desired gene.
  • This fragment is then ligated into a suitable plasmid, which it is possible to sequence and comprising restriction sites making it possible to easily extract the sequence inserted to introduce it into an expression vector and previously opened by compatible enzymes present in its multiple link site, for example by EcoRI and SmaI.
  • the ligation product can then be transfected into an appropriate host, for example an E. coli DH1 bacterial strain.
  • the purified plasmid comprises the expected nucleotide sequence of murine origin complementary to part C absent from the above human sequence from the same restriction site so as to allow phase ligation in the reading frame.
  • FIG. 1 represents the comparisons of the cDNAs of mouse C ⁇ 2 and human C ⁇ 1 chains of TCR.
  • FIG. 2 represents an oligonucleotide complementary to the cDNA coding for the C ⁇ 2 chain of the murine TCR except at the level of the two bases necessary for introducing the BglII site.
  • FIG. 3 represents an oligonucleotide specific for V ⁇ 17 chains of human TCR.
  • FIG. 4 represents the flow cytometry profiles obtained with an anti CD3 antibody from mice of DOIS19 transfectants called FRN17.4.14 (black profile) obtained with a chimeric DNA of the invention containing a human V ⁇ 17, relative to the line of departure DOIS19 (white profile).
  • Figure 5 shows the analysis of the V ⁇ 17 transfectant FRN 17.4.14 (black profiles) by the supernatant of E 175F3.15 (right histograms) and by the murine anti CD3 monoclonal (left histograms) compared to the starting line. DOIS 19 (white profiles).
  • FIG. 6 represents the double color labeling (anti CD3 of IMMUNOTECH labeled with phycoerythrin, E175F3.15 revealed by anti-mouse goat Ig labeled with FITC) of peripheral blood lymphocytes.
  • the sequence of the murine C ⁇ 2 chain is described in detail in Malissen M. et al (1984), Cell 37: 1101-1110.
  • a complete cDNA containing all the coding part of C ⁇ 2 was obtained from a cDNA library of the murine cell line KB5C20 (described in Albert et al (1982), Immunogenetics 16 : 533-549). This cDNA was cloned into the plasmid pUC19. The resulting plasmid pUCmC ⁇ 2 was transfected into the bacterial strain DH1.
  • the recombinant bacterium was deposited with the National Collection of Cultures of Microorganisms (CNCM) in Paris on February 12, 1991 under the n ° I-1032 and available without restriction.
  • the chosen site-directed mutagenesis procedure uses the filamentous phage M13mp18 BIORAD.
  • This bacteriophage is described precisely in Norrander et al (1983) Gene 26: 101-106. It is commercially available from several manufacturers, including BIORAD.
  • the principle of manipulation is as follows: the DNA fragment to be modified is cloned into the phage multiple binding site.
  • This phage vector exists in two forms during its life cycle.
  • the replicative form, in the bacterium is essentially double stranded, the encapsidated form is single stranded.
  • the DNA of the two forms is purifiable.
  • An oligonucleotide complementary to the region to be mutated, except for the desired mutation, is hybridized with the single-stranded form of the phage.
  • the complementary strand is then synthesized in vitro by a DNA polymerase, using the oligonucleotide containing the desired mutation to initiate the reaction.
  • a ligase is used to close the new strand on itself, at the 5 ′ end of the oligonucleotide.
  • the completely homologous double strand, except for the desired mutation is transfected into E. Coli resulting in two classes of mutant and non-mutant colonies. The mutant colonies are then selected.
  • this cDNA is cloned into phage M13mp18.
  • the oligonucleotide of FIG. 2 is used to carry out the mutagenesis.
  • This oligonucleotide is complementary to the cDNA coding for the murine C ⁇ 2 gene except for the two bases necessary to introduce the BglII site. These two bases are underlined in Figure 2.
  • the procedures for cloning, transfection, spreading, colony analyzes, purification of the single and double stranded forms for the phage M13mp18 are described precisely by Maniatis et al (already cited).
  • the bacterial host used is the E. coli strain JM101.
  • PUCmC ⁇ 2 is digested with Eco RI and Hind III. This digestion provides 3 fragments distinguishable by Agarose electrophoresis. The intermediate fragment of approximately 1000 base pairs (bp) is purified containing the entire coding sequence for murine C ⁇ 2. This fragment is ligated into the double-stranded DNA of the phage M13mp18 previously opened by Eco RI and HindIII, sites present in the multiple binding site of this vector. Colonies of E. coli JM 101 are then transfected.
  • the procedure used is precisely described in Maniatis et al. However, the following points should be made clear:
  • the oligonucleotide of FIG. 2 is synthesized on the Applied Biosystems 380B device, then purified on a sep Pak C18 column.
  • 0.2 ⁇ g of the single strand of M13EHC ⁇ 7 is hybridized with 3 pmol of OhC ⁇ kinase. No other oligonucleotide is used.
  • the initial temperature of the hybridization reaction is 70 ° C.
  • the polymerase used is T4 DNA polymerase (1 unit per reaction) to complete in double strand.
  • the transfection is carried out in the E. coli JM101 strain.
  • the selection of the mutant colonies is done by hybridization with the radiolabelled oligonucleotide of FIG. 2.
  • the hybridization temperature is 42 ° C. Two washings of 10 min at 4 ° C were sufficient to discriminate the colonies mutants by autoradiography.
  • the DNA of a subclone called muC ⁇ 215 from a colony identified by radioactive hybridization was digested with BglII, then sequenced by the Sanger method for confirmation of the desired mutation and integrity of the rest of the murine C ⁇ 2 sequence.
  • the double stranded DNA of this subclone is purified and used in the rest of the experiments.
  • p Bluescript SK+ is a fully characterized plasmid available commercially from the company STRATAGENE.
  • the double stranded DNA of muC ⁇ 215 is cut with EcoRI and EcoRV and the smaller of the two fragments purified on agarose gel. This fragment is ligated into p Bluescript previously opened by EcoRI and SmaI, two sites present in the multiple binding site of this plasmid, and transfected into the strain of E. coli DH1 serving as host for the chimera. Isolated colonies are analyzed by cleavage with two pairs of enzymes EcoRI-XbaI and EcoRI-BglII. A clone, called pBSmuC ⁇ 215, provides the expected bands (approximately 1.0 kb and approximately 0.5 kb) in electrophoresis. This plasmid is purified and used in the rest of the experiments.
  • lymphocytes from individuals Caucasians obtained from the Marseille Blood Transfusion Center. After Ficoll, the lymphocytes are adjusted to 1 million cells per milliliter in RPMI medium, 10% fetal calf serum 20 mM glutamine, 1 mM sodium pyruvate, 500 IU of penicillin, and stimulated with phytohemagglutinin A (10 ⁇ g / milliliter of final concentration) for 3 days. 200 million cells are used to purify their total RNA.
  • OhC ⁇ oligonucleotide specific for the two human chains C ⁇ 2 and C ⁇ 1 and containing the unique BglII site present on the gene segments C ⁇ 1 and C ⁇ 2 is synthesized in a manner analogous to OmC ⁇ .
  • This oligonucleotide is presented in FIG. 3.
  • a PCR reaction was carried out with the pair of oligonucleotides OhC ⁇ -OL17.
  • the quantity of starting RNA is 1 ⁇ g for the synthesis of the complementary strand of cDNA.
  • oligonucleotides OL17 and OhC ⁇ are used for the PCR reaction proper.
  • the reaction product is deposited on 1.4% Agarose gel for electrophoresis. After staining with ethidium bromide, a separate band of approximately 500 bp is cut and the DNA electroeluted. About 30 ng of DNA is obtained.
  • the chimeric genes are constructed by cloning the PCR fragment, cut by EcoRI and BglII, into the large fragment obtained from the digestion of pBSmuC ⁇ 215.
  • PBSmuC ⁇ 215 is cut with EcoRI and BglII and the large fragment called FpBSmuC ⁇ 215 is purified on Agarose gel and electroeluted.
  • 100 ng of the amplification product obtained with the oligonucleotides OL17, OhC ⁇ are cut with EcoRI and BglII.
  • the clone BSG ⁇ 17.4 is retained for the rest of the experiments.
  • This clone has the amino acid sequence V ⁇ 17 as described in Kimura et al (already cited). However, it has different D and J regions.
  • the TCR chain is a membrane protein which is expressed on the surface if, and only if, the other chains of the TCR complex associated with it (ie ⁇ chain and CD3 complex) are present.
  • DOIS19 developed by Letourneur F et al (1989) Eur J Immunol: 19: 2269-2274 is used.
  • DOIS19 is a mutant of a mouse T hybridoma which has all the components of TCR except the ⁇ chain. This line therefore does not express TCR on its surface.
  • the transfection of an exogenous ⁇ chain induces the expression of a functional receptor in the transfected cells.
  • the chimeric gene of the invention contained in BSG ⁇ 17.4 was transfected into this cell according to the protocols described below:
  • the expression vector chosen is the plasmid pH ⁇ Pr1-neo. It is described precisely in Gunning P et al (1987) Proc Natl Acad Sci USA; 84: 4831-4835.
  • the gene to be expressed is subcloned downstream of the ⁇ actin promoter and upstream of a polyadenylation site present on this plasmid. These two signals ensure the synthesis of a functional messenger RNA of the inserted gene.
  • this plasmid has the resistance gene neomycin which confers resistance to a series of antibiotics, in the preferred example the antibiotic G418, to the cells which have integrated the plasmid.
  • an origin of replication and an ampicillin resistance gene are also present allowing selection and replication in E coli.
  • the BSG ⁇ 17.4 plasmid is cut by the enzymes Bam HI and Sal I and the smaller of the two fragments (1.2 kb) is purified on Agarose gel.
  • This fragment comprising the entire coding sequence of the chimeric gene is ligated to the plasmid pH ⁇ PrI-neo previously opened with Bam HI and Sal I.
  • the ligation product is transfected into E. coli DH 1 and from the analysis of colonies isolated by the Bam HI and SalI cleavage, the clone called neo G ⁇ 17.4 is retained which provides the 1.2 kb fragment expected on the Agarose electrophoresis.
  • Néo G ⁇ 17.4 was deposited at the CNCM on September 24, 1991 under number I - 1144.
  • the transfection procedure chosen is electroporation.
  • the neo plasmid G ⁇ 17.4 is purified on a cesium chloride gradient as described in Maniatis et al (already cited), and taken up in TE (10 mM Tris, 1 mM EDTA pH8), at a rate of 1 mg / ml.
  • DOIS19 cells are cultured in complete medium (Dubelco modified Eagle medium, (DMEM) 10% of fetal calf serum (SVF), 2 mM glutamine, 1 mM sodium pyruvate, 500 IU penicillin, 500 IG streptomycin, 50 ⁇ M ⁇ mercaptoethanol).
  • the cells are then incubated for 24 h at 37 ° C. in a humid atmosphere at 7% CO2.
  • the cells are then distributed at a rate of 5.104 cells per ml in 24-well plates (Falcon) in complete medium supplemented with 3 mg / ml of antibiotics G418 (GIBCO).
  • the colonies resistant to the antibiotic are analyzed by flow cytometry.
  • the resistant clones FRN17.4.1 ... FRN17.4.30 are obtained.
  • 105 cells in a solution of DMEM 4% FCS, 0.02% sodium azide (FACS medium) and 15 ⁇ g / ml of murine anti CD3 antibody are incubated for 45 min at 4 ° C. with shaking.
  • FACS medium a polyclonal goat anti-rat immunoglobulin antibody labeled with FITC (Immunotech reagent at 15 ⁇ g / ml in FACS medium for 30 min at 4 ° C. with shaking. washes in FACS medium, the cells are fixed in PBS at 1% formaldehyde and analyzed on the Facscan flow cytometry apparatus of Becton Dickinson 105 DOIS19 cells are treated in the same way and serve as negative control.
  • mice The splenocytes of two immunized mice are fused with the myeloma X63 Ag 8.653 according to the protocol described by Kohler and Milstein (1975) Nature 256; 495-497.
  • the analysis of the supernatants of the clones resulting from the fusion is done by flow cytometry on FRN17.4.14 versus DOIS19. 100 ⁇ l of supernatant from each clone are removed. 50 ⁇ l are incubated with 50 ⁇ l of FACS medium containing 105 FRN17.4.14 cells. The remaining 50 ⁇ l are incubated with 50 ⁇ l of FACS medium but containing 105 DOIS19 cells. The protocol is also the same as that used for the analysis of transfectants.
  • FIG. 5 shows the profiles obtained on the transfectant FRN17.4.14 and DOIS19 with the supernatant E175F3.15 according to the protocol described for the screening of the antibodies compared to the profiles obtained with the murine anti CD3. It is noted that E175F3.15 marks in a comparable manner compared to the anti CD3 lines FRN17.4.14 and DOIS19.
  • transfectants FRN3.4.17 expressing a chimeric gene containing V ⁇ 3.1 were obtained; FRN8.1.2 expressing a chimeric gene carrying V ⁇ 8.1; FRN2.7.6 expressing a chimeric gene carrying V ⁇ 2.3 by transfecting the corresponding chimeric genes in DOIS19.
  • the supernatant E175F3.15 does not mark these different lines.
  • the antibodies recognize the V ⁇ 17 chain well, a subpopulation of peripheral T lymphocytes expressing V ⁇ 17 must be specifically labeled with these antibodies. Double labeling was carried out on peripheral blood with the antibody E175F3.15 revealed by a polyclonal labeled with FITC, and the antibody SPVT3b specific for human CD3 (reagent Immunotech) labeled with phycoerythrin.
  • the blood is taken from EDTA (10 mM final) to avoid clotting.
  • 100 ⁇ l of blood are centrifuged and the cell pellet is taken up in 200 ⁇ l of supernatant E175F3.15 and incubated for 30 min at room temperature.
  • Two washes are carried out in FACS medium and the cell pellet is taken up in 100 ⁇ l of FACS medium containing 15 ⁇ g / ml of a polyclonal goat anti-mouse immunoglobulin antibody (Immunotech reagent).
  • Two washes are carried out in the middle of FACS.
  • the cell pellet is taken up in FACS medium containing 15 ⁇ g / ml of a distinct mouse antibody (IgG2a control reagent from Immunotech).
  • the cells are analyzed on Facscan by Becton Dickinson.
  • the result obtained with the supernatant E175F3.15 is presented in FIG. 6. It can be seen that approximately 3% of the T cells are labeled with the antibody E175F3.15.
  • markings are carried out on the blood of other individuals using the antibody E175F3.15. This antibody marks between 1 and 3% of peripheral T cells for the individuals tested.
  • An injectable preparation comprising: - Monoclonal antibody of Example 2 5 mg - water for injections 1 ml

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EP92430022A 1991-09-26 1992-09-24 Molécules d'ADN recombinants contenant la partie codante d'une chaîne Vbêta17 du récepteur pour l'antigène des cellules T et leurs utilisations Withdrawn EP0534878A1 (fr)

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EP4211170A4 (en) * 2020-09-11 2024-10-09 Janssen Biotech, Inc. METHODS AND COMPOSITIONS FOR MODULATING BETA-CHAIN-MEDIATED IMMUNITY
US12264197B2 (en) 2019-03-11 2025-04-01 Janssen Biotech, Inc. Anti-Vβ17/anti-CD123 bispecific antibodies

Citations (2)

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Publication number Priority date Publication date Assignee Title
EP0340793A2 (en) * 1988-05-04 1989-11-08 Yeda Research And Development Company Limited Endowing cells with antibody specificity
WO1991010438A1 (en) * 1990-01-12 1991-07-25 Protein Design Labs, Inc. Soluble t-cell antigen receptor chimeric antigens

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0340793A2 (en) * 1988-05-04 1989-11-08 Yeda Research And Development Company Limited Endowing cells with antibody specificity
WO1991010438A1 (en) * 1990-01-12 1991-07-25 Protein Design Labs, Inc. Soluble t-cell antigen receptor chimeric antigens

Non-Patent Citations (2)

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Title
EUROPEAN JOURNAL OF IMMUNOLOGY vol. 17, 1987, VCH VERLAGSGESELLSCHAFT, DEUTSCHLAND pages 375 - 383 KIMURA, N. ET AL. 'Sequences and repertoire of the human T cell receptor alpha and beta chain variable region genes in thymocytes' *
IMMUNOLOGICAL REVIEWS no. 101, 1988, COPENHAGEN, DK pages 149 - 172 WILSON, R.K., LAI, E., CONCANNON, P., BARTH, R.K., HOOD, L.E. 'Structure, Organisation and polymorphism of murine and human T-cell receptor alpha and beta chain gene families.' *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US12264197B2 (en) 2019-03-11 2025-04-01 Janssen Biotech, Inc. Anti-Vβ17/anti-CD123 bispecific antibodies
EP4211170A4 (en) * 2020-09-11 2024-10-09 Janssen Biotech, Inc. METHODS AND COMPOSITIONS FOR MODULATING BETA-CHAIN-MEDIATED IMMUNITY

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